User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/14: Difference between revisions

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'''LCR Protocol Development'''<br>
'''LCR Protocol Development'''<br>
* Refer to entry [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/04/06 04/06/2015]
* Refer to entry [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/04/06 04/06/2015]
* Do PCR for all fragments with Phusion polymerase, use fresh dNTP's (from Rene)
* KAH
*
* '''MV10''' (5191 bp): Template - MV10 plasmid, F - MV10 f1, R - MV10 r1
* Gradient PCR to enhance 5 kb band
# 68°C
# 67.3°C
# 65.9°C
# 63.9°C
# 61.4°C
# 59.6°C
# 58.1°C
# 57°C
{| {{table}} cellspacing="3" <!-- PCR rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Rxn1-8
| bgcolor=#cfcfcf | (x9)
| rowspan="7" | Expected:<br>1. Gal4DB-mCh = 1170<br>3,4. ATF2 = 906<br>5,6. MV10 = 5191
| rowspan="7" | [[Image:somegel.jpg|200px|Hover name]]<br>10 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
| Template || 0.2 || 1.8
|-
| 10 uM fwd primer || 1.0 || 9.0
|-
| 10 uM rev primer || 1.0 || 9.0
|-
| 10 mM dNTPs || 1.0 || 9.0
|-
| Phusion pol. || 0.5 || 4.5
|-
| 5x HF buffer || 5.0 || 45.0
|-
| dH<sub>2</sub>O || 41.3 || 371.7
|-
| &nbsp; || 50.0
|}
Program: Phusion (block A) - edited to include 65°C - 55°C annealing gradient
* 98°C, 3 min
* 30x[98°C, 10 sec; 68°C - 57°C, 30 sec; 72°C, 2.5 min]
* 72°C, 10 min
* 4°C ∞
Conclusions
* tba




Revision as of 09:34, 14 April 2015

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04/14/15

  • LCR Development


LCR Protocol Development

  • Refer to entry 04/06/2015
  • Do PCR for all fragments with Phusion polymerase, use fresh dNTP's (from Rene)
  • KAH
  • MV10 (5191 bp): Template - MV10 plasmid, F - MV10 f1, R - MV10 r1
  • Gradient PCR to enhance 5 kb band
  1. 68°C
  2. 67.3°C
  3. 65.9°C
  4. 63.9°C
  5. 61.4°C
  6. 59.6°C
  7. 58.1°C
  8. 57°C
Reagent Rxn1-8 (x9) Expected:
1. Gal4DB-mCh = 1170
3,4. ATF2 = 906
5,6. MV10 = 5191 		Hover name
10 μL/lane, 1% agarose; Ladder
Template 0.2 1.8
10 uM fwd primer 1.0 9.0
10 uM rev primer 1.0 9.0
10 mM dNTPs 1.0 9.0
Phusion pol. 0.5 4.5
5x HF buffer 5.0 45.0
dH2O 41.3 371.7
  50.0

Program: Phusion (block A) - edited to include 65°C - 55°C annealing gradient

  • 98°C, 3 min
  • 30x[98°C, 10 sec; 68°C - 57°C, 30 sec; 72°C, 2.5 min]
  • 72°C, 10 min
  • 4°C ∞


Conclusions

  • tba






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